Codon-optimized nucleic acid for coding apo-clytin-II and method for using the same

ABSTRACT

A codon-optimization of nucleic acid for coding apo-clytin-II protein is provided. Luminescent activity of clytin-II is remarkably enhanced. Accordingly, compared with the conventional use of the wild-type clytin-II, an intracellular calcium ion can be detected much more easily.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of Japan application serialno. 2009-018339, filed on Jan. 29, 2009. The entirety of theabove-mentioned patent application is hereby incorporated by referenceherein and made a part of specification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to a codon-optimized nucleicacid for coding apo-clytin-II protein, apo-clytin-II protein, and methodfor preparing the same.

2. Description of Related Art

In the luminescent jellyfish of coelenterates, six photoproteins are nowknown to emit light by specifically binding to calcium ions. Among theseproteins, aequorin and obelin have been studied in detail. Thesecalcium-binding photoproteins have been used as a biological calciumindicator and for determining calcium concentration in cells.Furthermore, these proteins have been used in the following process. Byusing the gene of the protein, information about the important modifierand the biological mechanism in the cellular environment that aredependent upon calcium is deduced.

The photoprotein clytin was isolated from other luminous jellyfish,Clytia gregaria, in 1982 (see Non-patent Reference 1). Its gene wasisolated in 1993, and it was confirmed that the gene could be expressedin E. coli (see Non-patent Reference 2). Thereafter, gene analysis wasperformed in detail by Inouye et al., and new gene was identified as newclytin with different luminescence property. The new gene from Clytiagregaria was named as clytin-II to distinguish from the previouslyisolated clytin (=clytin-I) (see Non-patent Reference 3). The clytin-IIgene can be expressed in E. coli, and can be purified as recombinantclytin-II, which exhibits a potential application in the determinationof a biological calcium indicator or the calcium concentration.

Compared with aequorin and clytin-I, the luminescence capacity of thetotal light emission per protein of clytin-II is almost the same;however, the initial luminescence intensity of clytin-II is about fivetimes higher. Thus, the S/N ratio of clytin-II is five times higher thanthat of aequorin and clytin-I, which indicates that clytin-II may besuperior to aequorin and clytin-I when used in a detection system.

Clytin-II having the luminescence ability is a complex containingapo-clytin-II protein with a molecular weight of about 22,000 andcoelenterazine binding molecular oxygen. A molecule of the clytin-IIprotein has three sequences that can bind calcium ion. When clytin-II isbound to calcium ion, blue light (λmax=470 nm) is emitted, and carbondioxide and coelenteramide are generated. Since the luminescenceintensity is dependent on the concentration of calcium ion, clytin-IIcan be used to determine the calcium concentration with highsensitivity.

According to the prior art, the amino acid sequence of the photoproteinclytin-II is initially published by Inouye (see Non-patent Reference 3),and is disclosed in the EMBL sequence database under accession numberAB360785 (disclosed as SEQ ID NO: 1 in this specification).

Amino acid sequence identity between clytin-II and other photoproteins,clytin-I, aequorin, obelin, and mitrocomin, is 88.4%, 61.9%, 76.2%,60.8% respectively, while the identity with aequorin, obelin, andmitrocomin is low (see Non-patent Reference 3).

Furthermore, in Japanese Patent Publication 2005-506053, acodon-optimized (humanized) gene sequence based on the amino acidsequence of the photoprotein aequorin is reported. The amino acidsequence homology of codon-optimized aequorin with clytin-II shows 61.9%identity, and the luminescence pattern is different from clytin-II.Further, the nucleotide sequence identity between aequorin and clytin-IIis 64.6%, so aequorin and clytin-II can be identified as totallydifferent functional molecules.

PRIOR ART REFERENCES

[Patent Reference]

[Patent Reference 1] Japanese Patent Publication 2005-506053

[Non-patent References]

[Non-patent Reference 1] Comp. Biochem. Physiol. (1982) 72B, 77-85

[Non-patent Reference 2] FEBS Lett. (1993) 333, 301-305

[Non-patent Reference 3] J. Biochem. 143 (2008) 711-717

SUMMARY OF THE INVENTION

Currently there is no actual evidence on stable expression ofrecombinant clytin-II in eukaryotic cells or a host assay cell. There isalso no actual result on the use of clytin-II as a reporter protein fordetecting the change of intracellular calcium concentration.Furthermore, there is no study on the usefulness of clytin-II in thescreening of small molecules that stimulate G protein-coupled receptorand/or ion channel. Moreover, there is no report on the optimizedclytin-II sequence.

When analyzing the change of intracellular calcium concentration inmammalian cells with clytin-II, it is necessary to introduce a plasmidcontaining a nucleic acid for coding apo-clytin-II into cells. Theintroduction of nucleic acids into cells can be achieved by thefollowing standard transfection technology: formation of DNA complexwith ionic lipid reagent, or precipitation with CaPO₄.

However, in the transformed mammalian cells, when merely a small amountof cell groups contain the apo-clytin-II expressing plasmid, theexpression level of apo-clytin-II protein is low. As a result, in acalcium analysis using clytin-II regenerated from apo-clytin-II, theapplication of clytin-II is limited due to low luminescence intensity(low light emission). The inventors of the present invention set forththe following hypothesis based on this fact: the increase in theintracellular expression of apo-clytin-II protein can effectivelyachieve higher light emission and signal to noise ratio in a calciumanalysis.

Furthermore, the inventors of the present invention focus on thefollowing aspects. The increase of apo-clytin-II gene transcriptionallows the performance of the calcium analysis in a mammalian cell,which is difficult to achieve with the standard transduction technology.The amplification can be achieved by introducing apo-clytin-II gene viathe retrovirus gene delivery method, or other methods in which thenumber of transcribed genes is generally less than that of transientlytransduced genes. Further, the gene delivery to a wider range ofmammalian cells can be achieved.

Moreover, the inventors of the present invention have not identify anypublication regarding a codon-optimized version with increasedexpression on apo-clytin-II protein in mammalian cells, and the use ofthe same.

Therefore, the inventors of the present invention have conductedextensive researches on the problems above in the prior art. As aresult, the inventors have found that codon optimization (hereinafterknown as humanization) of a nucleic acid for coding apo-clytin-IIprotein can significantly improve the luminescent activity of clytin-II;thus, the detection of intracellular calcium ion is easier, comparedwith that using wild-type clytin-II in the prior art. The inventors havecompleted the invention based on the above-observations.

The present invention includes at least the following features.

(1) A codon-optimized nucleic acid is for coding apo-clytin-II protein.

(2) The codon-optimized nucleic acid according to Item 1 is for codingapo-clytin-II protein having an amino acid sequence of SEQ ID NO: 1, ora variant thereof.

(3) A codon-optimized nucleic acid is formed by fusing thecodon-optimized nucleic acid according to Item 1 or 2 with a nucleicacid for coding other proteins.

(4) The codon-optimized nucleic acid according to any one of Items 1 to3 includes one or more codon selected from the group consisting of codonfor coding CGC arginine, codon for coding AAC asparagine, codon forcoding GAC aspartic acid, codon for coding CAG glutamine, codon forcoding GAG glutamic acid, codon for coding GGC glycine, codon for codingCAC histidine, codon for coding ATC isoleucine, codon for coding CTGleucine, codon for coding AAG lysine, codon for coding CCC proline,codon for coding TTC phenylalanine, codon for coding TCC or AGC serine,codon for coding TAC tyrosine, and codon for coding GTG valine, and thenumber of the one or more codon is greater than that of a nucleic acidfor coding wild-type jellyfish apo-clytin-II protein of SEQ ID NO: 2.

(5) An apo-clytin-II protein is expressed by the codon-optimized nucleicacid according to any one of Items 1 to 4.

(6) A clytin-II includes the apo-clytin-II of Item 5 as a component.

(7) An expression vector, wherein the position of its codon-optimizednucleic acid for coding apo-clytin-II protein is under the regulatorycontrol of a promoter that functions in mammalian cells.

(8) The expression vector according to Item 7 includes thecodon-optimized nucleic acid for coding apo-clytin-II protein, and aregulatory sequence for controlling the expression of thecodon-optimized nucleic acid for coding apo-clytin-II protein inmammalian cells.

(9) A recombinant host cell includes a codon-optimized nucleic acid forcoding apo-clytin-II protein.

(10) In the recombinant host cell according to Item 9, the host cell isa mammalian cell.

(11) In the recombinant host cell according to Item 9, the host cell isa human cell.

(12) In the recombinant host cell according to Item 9, the host cell isa non-human mammalian cell.

(13) A method for preparing apo-clytin-II protein includes the steps of:

(i) preparing a recombinant expression vector that allows acodon-optimized nucleic acid for coding apo-clytin-II protein to beunder the regulatory control of a promoter that can function in a hostcell;

(ii) introducing the recombinant expression vector into the host cell;and

(iii) culturing the host cell, for expressing the apo-clytin-II protein.

(14) A method for enhancing the luminescence intensity of clytin-IIincludes combining a codon-optimized nucleic acid for codingapo-clytin-II protein with a regulatory nucleic acid for expressing thecodon-optimized nucleic acid to form a nucleic acid, and introducing theresulting nucleic acid into a host cell.

(15) An application of a codon-optimized nucleic acid for codingapo-clytin-II protein, for enhancing the luminescence intensity ofclytin-II in host cells.

(16) A method for determining the capability of a compound forinhibiting a receptor through the change of intracellular calcium ionupon activation includes contacting a host cell genetically engineeredto express apo-clytin-II protein with coelenterazine or a derivative ofcoelenterazine, and then determining the amount of the generated light.

(17) The method according to Item 16 further includes recording thecapability of inhibiting the receptor.

EFFECT OF THE INVENTION

The codon-optimized nucleic acid for coding apo-clytin-II is a novelsubstance, and the humanized apo-clytin-II protein (hereinaftersometimes known as “codon-optimized apo-clytin-II protein”) obtained byintroducing the codon-optimized nucleic acid into a host cell exhibits ahigher luminescent activity when forming a complex (humanized clytin-II)consisting of the luminescent substrate and molecular oxygen, comparedwith that using wild-type apo-clytin-II. Furthermore, the clytin-IIobtained by the present invention is very effective in manyapplications, such as the detection of intracellular calcium ion.

In order to make the features and advantages of the present inventioncomprehensible, specific embodiments accompanied with figures aredescribed in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention, and are incorporated in and constitute apart of this specification. The drawings illustrate embodiments of theinvention and, together with the description, serve to explain theprinciples of the invention.

FIG. 1 is a schematic diagram of the codon-optimized apo-clytin-IIprotein expression vector pCMX-hCLII in eukaryotic organisms used in thepresent invention.

FIG. 2 is a schematic diagram of the codon-optimized apo-clytin-IIprotein expression vector piP-H-hCLII in prokaryotic organisms used inthe present invention.

DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to the present embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers are used in thedrawings and the description to refer to the same or like parts.

The first aspect of the present invention is a codon-optimized nucleicacid for coding apo-clytin-II protein (hereinafter also referred ascodon-optimized apo-clytin-II nucleic acid).

In preferred embodiments, the codon-optimized apo-clytin-II proteincontains a sequence of SEQ ID NO: 1 or a truncated version thereof.

The truncated version is a protein in which one or more amino acids onor near the N-terminus or C-terminus are removed. In an embodiment, thetruncated version is a protein with less than 50 amino acids beingremoved from the C-terminus. Herein, the truncated version is requiredto maintain several luminescence properties.

Another embodiment includes variants of apo-clytin-II protein ofwild-type apo-protein disclosed by Inouye (J. Biochem. 143 (2008)711-717). Herein, the variants include enhanced or modified luminescenceproperty. Further, the variants are preferably amino acid substitutionsbased on the sequence expressed by SEQ ID NO: 1, or variants obtainedfrom the wild-type amino acid sequence by changing one, two, or threeamino acids. Examples of variants of the apo-clytin-II protein mayinclude variant with valine at position 62 of SEQ ID NO 1 replaced byisoleucine, variant with alanine at position 78 replaced by proline, andvariant with glutamic acid at position 91 replaced by lysine, and thelike.

Furthermore, the sequence identity of variation sequences in theembodiments of the present invention with the sequence of SEQ ID NO: 1is preferably 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, and more preferably 99% or more. The sequenceidentity between two sequences can be evaluated by, for example, bestcomputer alignment analysis with the appropriate software, such as NCBIBlast, WashU Blast2, Fasta, and PILEUP, or using a scoring matrix likeBlosum62. Herein, the sequence identity between two sequences isdetermined by finding an approximation of the “gold-standard” alignmentalgorithm of Smith-Waterman.

The terms “codon-optimization” and “humanization” used in thespecification refer to one or more replacement of codon of jellyfishapo-clytin-II with codon more frequently used in human genes, preferablya significant number of replacement, to adapt to the expression inmammalian cells, especially in human cells.

In another preferred embodiment, the percentage of humanized codon ispreferably 10% or more, 20% or more, 30% or more, 40% or more, 50% ormore, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more,98% or more, and more preferably 99% or more.

The codon-optimized nucleic acid of the present invention is generallycDNA, and can also contain genome copy.

The codon-optimized nucleic acid can be chemically synthesized by thestandard techniques in the art, or obtained by mutation of wild-typegenome or wild-type cDNA. Nucleotide variation or nucleotide mutationcan be achieved by novel polynucleotide synthesis, polymerase chainreaction (PCR), site-directed mutagenesis using an appropriatelydesigned oligonucleotide primer, or other methods known by those ofordinary skill in the art. The codon-optimized apo-clytin-II nucleicacid of the present invention can be designed to have an appropriaterestriction enzyme recognition sequence at the terminus, or arestriction enzyme sequence with an additional base sequence can be usedto facilitate the cloning of the humanized gene on a plasmid vector.

The codon-optimized apo-clytin-II protein-coding amino acid sequence canbe expressed by various expression vector/host systems. The expressionvector/host systems are not specifically limited, and include, forexample, recombinant adeno-virus system, adeno-associated virus (AAV)system, or retrovirus system. The expression vectors useful in thepresent invention preferably include, for example, vaccinia virus,cytomegalo virus, herpes simplex virus, and defective hepatitis B virus.Herein, in order to express the codon-optimized apo-clytinII nucleicacid, a mammalian expression system is preferably used; however, othervectors and host cells, such as bacteria, yeast, plants, fungi andinsects can also be used.

Expression vectors in a mammalian expression system generally include anorigin of replication, a promoter, a transcription initiation site,optionally a signal peptide, a polyadenylation site, and a transcriptiontermination site, and the like. The vectors generally also contain oneor more antibiotics-resistant marker(s) for selection. An appropriateexpression vector in the present invention may be plasmid, cosmid,phage, or virus, such as retrovirus. The coding sequence of the proteinis placed under the control of an appropriate promoter (i.e., HSV, CMV,TK, RSV, SV40, etc.), regulatory factors, and transcription terminator,such that the nucleic acid coding the protein (hereinafter sometimescalled “coding sequence”) is transcribed into RNA in the host celltransformed or transduced with the expression vector.

The coding sequence may or may not contain a signal peptide or leadersequence for the secretion of the target protein out of the host cell. Avector useful in the present invention preferably contains at least onemultiple cloning site. In an embodiment, there is a cloning site or amultiple cloning site situated between the promoter and thecodon-optimized apo-clytin-II nucleic acid. The cloning site is in-framesince it is close to the codon-optimized apo-clytin-II nucleic acid; andthus, it can be used to generate an N-terminus fusion protein by cloninga second nucleic acid sequence at the cloning site. In anotherembodiment, there may be a cloning site or a multiple cloning sitesituated immediately downstream of the codon-optimized apo-clytin-IInucleic acid to facilitate the creation of C-terminus fusions in asimilar fashion to that for N-terminus fusions described above.

The expression and purification of the codon-optimized apo-clytin-IIprotein of the present invention can be easily accomplished by themethods well-known in the art (for example, those described in“Molecular Cloning-A Laboratory Manual, second edition 1989” by Sambrooket al.). The use of expression vectors and plasmids is well-known tothose of ordinary skill in the art. In almost all cases, any mammaliancell expression vector can be used in the expression of thecodon-optimized apo-clytin-II protein of the present invention.

According to another aspect of the present invention, an expressionvector containing a regulatory sequence capable of controlling theexpression of a codon-optimized apo-clytin-II nucleic acid in host cellsis provided.

In the expression vector of the present invention, the codon-optimizedapo-clytin-II nucleic acid is preferably under the transcriptionregulation of a promoter that can function in mammalian cells.

The vectors containing a codon-optimized apo-clytin-II nucleic acid canbe introduced, that is being transformed or transduced, into mammaliancells such as Chinese hamster ovary (CHO) cell, bacteria such as E.coli, yeasts such as Saccharomyces cerevisiae or Pichia pastoris, orother suitable hosts that can easily be subjected to operations such asmutagenesis, cloning, or expression. The implementation of the presentinvention is not dependent on any particular host cell line or vector,and is not limited thereby. The host cells or vectors useful in thepresent invention are known and can be selected by those of ordinaryskill in the art.

The method for introducing vectors into cells is preferably thosecommonly used, and includes, for example, calcium phosphate method,electroporation method, microinjection method, diethylaminoethyl-dextranmethod (DEAE-dextran method), method using a liposome reagent,lipofection method using a cationic lipid. In the case of a cyclicvector, the vector can be linearized by a known method and thenintroduced into cells.

A host cell transformed or transduced with a vector containing acodon-optimized apo-clytin-II nucleic acid can be cultured under theconditions suitable for the expression of the target substance and therecovery of the target substance from cell culture. The expressedcodon-optimized apo-clytin-II protein may be secreted into the cultureor stored in the cell, depending on the sequences used, which is basedon whether a suitable secretion signal sequence is present or not. Here,both transiently transformed cells/cell lines and stably transformedcells/cell lines are considered as objects of the research.

Suitable host cells for expressing the codon-optimized apo-clytin-IIprotein include, for example, CHO, COS, HeLa, BHK, Vero, MDCK, HepG2,HEK293, K562, etc.

In making a transgenic animal capable of expressing the codon-optimizedapo-clytin-II protein, for example, an expression system disclosed inU.S. Pat. No. 5,714,666 can be used.

The present invention further provides a transgenic non-human animal,which contains a codon-optimized apo-clytin-II nucleic acid and aregulatory sequence for controlling the expression of thecodon-optimized apo-clytin-II protein in the cell.

The transgenic animal is not particularly limited, and is preferably amouse in the present invention.

In another aspect of the present invention, a host cell suitable forexpressing the codon-optimized apo-clytin-II protein of the presentinvention is provided, which is a recombinant host cell containing acodon-optimized apo-clytin-II nucleic acid. According to the presentinvention, the host cell is not limited, and preferably is a mammaliancell. The mammalian cells include human cells and non-human cells, suchas CHO-K1 and Phoenix cells. Among the mammalian cells, human cells aremost preferred in the present invention.

The recombinant host cell according to an embodiment of the presentinvention generates the codon-optimized apo-clytin-II protein in asufficient amount that can be detected when forming the clytin-II.

According to the codon usage frequency formed from the coding sequenceof the wild-type clytin-II as shown in Table 1, the apo-clytin-II codonof the wild-type jellyfish at position 3 is preferably either A or U.Generally speaking, the residue at position 3 of the preferred mammaliancodon is preferably C or G, and most preferably C, regardless thehomology of the two residues at position 1 and position 2. Haas et al.,(Current Biology. 6(3):3135-324, 1996) analyzed the comparison of 100high-expression human genes, and the results showed the followingtendencies. For example, 53% of the alanine (GCX) residue in thehigh-expression genes is coded by GCC, 17% is coded by GCT, 13% is codedby GCA, and 17% is coded by GCG. Similarly, the serine residue has thefollowing statistics: TCC (28%), TCT (13%), TCA (5%), TCG (9%), AGC(34%), and AGT (10%). As shown in the table of the usage frequency ofthe codon among jellyfish and human being, in the amino acids with onlytwo codons for selection, the wild-type jellyfish apo-clytin-II nucleicacid generally uses the most non-preferred codon compared to thepreferred codon of human genes.

TABLE 1 Usage frequency of wild-type clytin-II codon  [Table 1] 1^(st)2^(nd) base 3^(rd) base U C A G base U TTT Phe 6 TCT Ser 3 TAT Tyr 2 TGTCys 3 T TTC 7 TCC 1 TAC 2 TGC end 0 C TTA Leu 3 TCA 3 TAA end 0 TGA Trp0 A TTG 6 TCG 0 TAG 0 TGG 6 G C CTT 2 CCT Pro 1 CAT His 2 CGT Arg 2 TCTC 2 CCC 1 CAC 3 CGC 0 C CTA 0 CCA 5 CAA Gln 2 CGA 1 A CTG 1 CCG 0 CAG2 CGG 0 G A ATT Ile 3 ACT Thr 1 AAT Asn 3 AGT Ser 4 T ATC 7 ACC 2 AAC 5AGC 0 C ATA 1 ACA 4 AAA Lys 13 AGA Arg 1 A ATG Met 4 ACG 1 AAG 4 AGG 1 GG GTT Val 5 GCT Ala 6 GAT Asp 12 GGT Gly 3 T GTC 2 GCC 1 GAC 11 GGC 5 CGTA 0 GCA 4 GAA Glu 11 GGA 4 A GTG 0 GCG 1 GAG 3 GGG 1 G

In constructing a codon-optimized apo-clytin-II nucleic acid, each codonis replaced by either C or G at the third position; thus, each codon isconverted into mammalian equivalents. If this cannot be accomplished, itis possible that a problematic restriction enzyme site is introduced,and accordingly, the next frequently used nucleotide in thehigh-expression human genes can be used.

Preferably, the number of the codons for coding GCC alanine is increasedin the codon-optimized apo-clytin-II nucleic acid of present invention,compared with in the wild-type jellyfish nucleic acid of SEQ ID NO: 2.

Herein, the so-called “increase in the number of codons” does not meanthat there are more alanine amino acids than codon-optimized type;instead, it means that the relative number of codons for coding GCCalanine is greater than that of the codon-optimized apo-clytin-IInucleic acid. However, one can not exclude that there is a greaternumber of specific amino acids in the specific codon-optimizedapo-clytin-II protein than that in the wild-type protein. For example,the codon-optimized apo-clytin-II protein can have a relatively greaternumber of amino acids, compared with the wild-type protein sequence ofSEQ ID NO: 2.

By reference to the above definition, the codon-optimized apo-clytin-IInucleic acid of the present invention preferably includes one codonselected from the group consisting of codon for coding CGC arginine,codon for coding AAC asparagine, codon for coding GAC aspartic acid,codon for coding CAG glutamine, codon for coding GAG glutamic acid,codon for coding GGC glycine, codon for coding CAC histidine, codon forcoding ATC isoleucine, codon for coding CTG leucine, codon for codingAAG lysine, codon for coding CCC proline, codon for coding TTCphenylalanine, codon for coding TCC or AGC serine, codon for coding TACtyrosine, and codon for coding GTG valine, and the number of the onecodon is greater than that of the wild-type jellyfish apo-clytin-IInucleic acid of SEQ ID NO: 2.

The human optimized apo-clytin-II with 7 amino acids added at the aminoterminus is one of the preferred codon-optimized apo-clytin-II nucleicacids in the present invention. The codon-optimized nucleic acid of thepresent invention can be formed by fusing a codon-optimizedapo-clytin-II nucleic acid and a nucleic acid for coding other proteins.As a result, the fusion protein can be expressed in the host cell havinga control sequence for expressing the protein and the above sequenceformed by the fusion. Furthermore, this result can be obtained for theN-terminus fusion protein or the C-terminus fusion protein.

The other proteins fused with the codon-optimized apo-clytin-II proteinof the present invention are not particularly limited, and include, forexample, secretion or other control sequence, tag sequence (e.g., 6-histag), targeting sequence, and a green fluorescent protein that functionsas a reporter protein. Additionally, HA 1 epitope, which functions as arecognition sequence, can be included, so that the expression and thedetermination of the concentration of apo-clytin-II protein can beachieved.

Another aspect of the present invention is a method for preparing aapo-clytin-II protein, which includes the steps of:

(i) preparing a recombinant expression vector that allows acodon-optimized apo-clytin-II nucleic acid to be under the control of apromoter that can function in host cells;

(ii) introducing the recombinant expression vector into an appropriatehost cell; and

(iii) allowing the host cell to express the codon-optimizedapo-clytin-II protein.

In the preparation method of the present invention, the host cell is notparticularly limited, and preferably a mammalian cell, in which case,the promoter is preferably a promoter that can function in mammals hostcell.

The method of introducing the recombinant expression vector into thehost cell is not particularly limited, and can be the methods describedabove or below. Furthermore, the method of expressing thecodon-optimized apo-clytin-II protein is also not particularly limited,and includes, for example, culturing the host cell under the conditionssuitable for expressing the codon-optimized apo-clytin-II protein.

Furthermore, the preparation method of the present invention can furtherinclude the step of:

(iv) purifying the expressed codon-optimized apo-clytin-II protein froma significant amount of other intracellular proteins.

After the step (iv), the purity of the codon-optimized apo-clytin-IIprotein is preferably 70% or more, 85% or more, and more preferably 95%or more.

Another aspect of the present invention is a method of enhancing theluminescence intensity of clytin-II, and the method includes introducinga nucleic acid into a host cell, in which the nucleic acid is formed byfunctionally combining a codon-optimized nucleic acid that codes andgenerates the apo-clytin-II protein with a control sequence that canachieve the effective expression of the nucleic acid.

In preferred embodiments, the nucleic acid containing thecodon-optimized apo-clytin-II nucleic acid functionally combined withthe control sequence is introduced into the host cell by means oftransduction, transformation, or electroporation, etc. Further, the hostcell is cultured under the conditions suitable for expressing thecodon-optimized apo-clytin-II protein. “Enhancement of the luminescenceintensity of clytin-II” refers herein to the increase in theluminescence intensity compared with the same expression system, exceptthat the wild-type (non-humanized) jellyfish apo-aequorin gene is used.

Another aspect of the present invention is an application of acodon-optimized apo-clytin-II nucleic acid for enhancing theluminescence intensity of clytin-II in host cells.

Another aspect of the present invention is a method of determining thecapability of a compound to block, inhibit, or antagonize a receptorupon activation, such as G-protein coupled receptor (hereinaftersometimes called GPCR) or ion channel, by the change of intracellularcalcium ion. For example, GPCR can be expressed in a cell line, such asHEK293 cell or CHO cell, which is genetically engineered to express thecodon-optimized apo-clytin-II protein. Before determination, the cellsare incubated in the presence of coelenterazine; the generatedapo-clytin-II thereby can be converted into clytin-II.

Specifically, the test compound is added into the cell, followed byadding a ligand. Further, the light emission generated by the increasedcalcium ion due to the activation of the receptor is determined with astandard luminometer. Then, in estimating the degree of inhibition bythe test compound, the luminescence intensity from the cell treated bythe compound is compared with the luminescence intensity from the celltreated by the ligand alone. Additionally, the clytin-II cell is treatedby the test compound, and the amount of light emission is directlydetermined, so as to directly locate the receptor agonist.

Therefore, according to the aspect of the present invention, thereceptor contained in the regulation of intracellular calcium and themammalian cells operated in the manner that the clytin-II from thehumanized gene is expressed are incubated with the test compound. Then,a coelenterazine cofactor is added, and the luminescence intensity isdetermined. The luminescence intensity herein is an indicator of thelevel of intracellular calcium that is released. Furthermore, the testresult, that is, the capability of the test compound to inhibit orregulate the receptor can be recorded, for example, on paper or byelectronic means.

The SEQ ID NOs: in the sequence list of the specification represent thefollowing sequences, respectively.

[SEQ ID NO: 1] represents the amino acid sequence of the wild-typeapo-clytin-II.

[SEQ ID NO: 2] represents the base sequence of the wild-typeapo-clytin-II.

[SEQ ID NO: 3] represents the base sequence of the codon-optimizedapo-clytin-II.

[SEQ ID NO: 4] represents the base sequence of the human-type clytingene inserted into the expression vector pCMX-hCLII fabricated inExample 1.

[SEQ ID NO: 5] represents the amino acid sequence of the human-typeclytin gene inserted into the expression vector pCMX-hCLII fabricated inExample 1.

[SEQ ID NO: 6] represents the base sequence of the human-type clytingene inserted into the expression vector piP-H-hCLII fabricated inExample 2.

[SEQ ID NO: 7] represents the amino acid sequence of the human-typeclytin gene inserted into the expression vector piP-H-hCLII fabricatedin Example 2.

[SEQ ID NO: 8] represents the base sequence of the primer used inExample 2.

[SEQ ID NO: 9] represents the base sequence of the primer used inExample 2.

[SEQ ID NO: 10] represents the base sequence of the primer used inExample 2.

[SEQ ID NO: 11] represents the base sequence of the primer used inExample 2.

[SEQ ID NO: 12] represents the base sequence of the primer used inExample 2.

[SEQ ID NO: 13] represents the base sequence of the primer used inExample 2.

EXAMPLES

Hereinafter, the present invention is described in detail with theexamples, but not limited to the examples.

Example 1 Design and Chemical Synthesis of the Codon-Optimizedapo-clytin-II Nucleic Acid

The codon-optimized apo-clytin-II nucleic acid is designed in thefollowing manner (Sequence list 3): without changing the amino acidsequence of apo-clytin-II, using the amino acid codon frequently used inhuman body, and converting the transcription factor recognition sequenceinto a non-activated sequence, removing the site that may be subjectedto splicing, eliminating six restriction enzyme sites for baserecognition, and without forming a palindrome sequence to make a loopstructure. The codon usage frequency of the codon-optimizedapo-clytin-II is shown in Table 2. Compared with the codon frequency ofwild-type apo-clytin-II in Table 1, the codon-optimized apo-clytin-IInucleic acid is obviously an optimized design based on the humanizedcodon.

The optimized design codon-optimized apo-clytin-II nucleic acid of issynthesized by routine method and chemical synthesis. The cloning isperformed in pBlueScript SK(+) (Stratagene Company) at the EcoRI/SalIrestriction enzyme site to construct the pBlue-hCLII plasmid. Thecodon-optimized apo-clytin-II nucleic acid is confirmed by determiningthe base sequence with a DNA sequencer (manufactured by AppliedBiosystems, ABI).

TABLE 2 Codon usage frequency of the codon-optimized clytin-II [Table 2] 1^(st) 2^(nd) base 3^(rd) base U C A G base U TTTPhe 0 TCT Ser 0 TAT Tyr 0 TGT Cys 0 T TTC 13 TCC 0 TAC 4 TGC 3 C TTA Leu0 TCA 0 TAA end 0 TGA end 0 A TTG 0 TCG 0 TAG 0 TGG Trp 6 G C CTT 0 CCTPro 0 CAT His 0 CGT Arg 0 T CTC 0 CCC 7 CAC 5 CGC 0 C CTA 0 CCA 0 CAAGln 0 CGA 0 A CTG 14 CCG 0 CAG 4 CGG 0 G A ATT Ile 0 ACT Thr 0 AAT Asn 0AGT Ser 0 T ATC 11 ACC 8 AAC 8 AGC 11 C ATA 0 ACA 0 AAA Lys 0 AGA Arg 5A ATG Met 4 ACG 0 AAG 17 AGG 0 G G GTT Val 0 GCT Ala 0 GAT Asp 0 GGT Gly0 T GTC 1 GCC 12 GAC 23 GGC 13 C GTA 0 GCA 0 GAA Glu 0 GGA 0 A GTG 6 GCG0 GAG 14 GGG 0 G

Example 2 Construction of the Codon-Optimized apo-clytin-II ProteinExpression Vector in Cultured Animal Cells

The construction of the codon-optimized apo-clytin-II protein expressionvector is as follows. Firstly, a novel expression vector pCMX-Linker isconstructed in cultured animal cells. Specifically, in pCMX-GFP(described in Ogawa et al., (1995) PNAS. 92(25), 11899-11903), linkersLinker F(5)A-Sal (5′ GT ACC ACC ATG CTC GAG CTG CAG GAA TTC TCT AGA G3′) (SEQ ID NO: 7) and Linker R(5)A-Sal (5′ TC GAC TCT AGA GAA TTC CTGCAG CTC GAG CAT GGT G 3′) (SEQ ID NO: 8) having a chemical synthesizedmultiple cloning sequence is inserted at the restriction enzyme site,i.e., the Asp718/SalI site, to construct the novel expression vectorpCMX-Linker. That is, the novel expression vector is controlled by theCMV promoter, which has the Kozak sequence and multiple cloning sitesequence (Asp71811 XhoI/PstI/EcoRI/XbaI/SalI/EcoRV/BamHI/MscI/NheI) inthe downstream.

Thereafter, the codon-optimized apo-clytin-II protein expression vectorusing the novel expression vector pCMX-Linker is constructed as follows.

After digestion with the restriction enzyme EcoRI/SalI, with the routinemethod, pBlue-hCLII is linked to the EcoRI-SalI site of the pCMX-Linker,and the expression vector pCMX-hCLII is constructed as shown in FIG. 1.Further, the base sequence is determined by a DNA sequencer(manufactured by Applied Biosystems, ABI) to confirm the inserted DNA.Additionally, the control vector containing the wild-type apo-clytinnucleic acid as a control of the codon-optimized apo-clytin-II nucleicacid, i.e., pCMX-CLII, is constructed by the same method.

Example 3 Introduction of the Vector into Cells

(1) Truncation and Purification of Expression Plasmid

The pCMX-hCLII plasmid obtained in Example 2 is used to perform thefollowing experiments. The pCMX-hCLII plasmid is purified in E. coliJM83 by using Endofree Plasmid Maxi kit (manufactured by QIAGENCompany), and then dissolved into sterilized water to a concentration of1 μg/μl. Similarly, pCMX-hCLII and the firefly luciferase vector(pGL-control: Promega Company) applied as internal standard are used.

(2) Transfection

The cell line from human cervical cancer, i.e., HeLa line, is culturedwith DMEM medium (with high-concentration glucose, Wako Chemicals)containing 10% fetal bovine serum (Invitrogen Company), and seeded in6-well culture dish in 2×10⁵ cells/well, and cultured in an incubatorunder the condition of 37° C. and 5% CO₂. After 24 hours, by using aFuGene HD transfection kit (Roche Company), the purified pCMX-hCLIIplasmid is transfected into the HeLa cell in order to be used in theexperiment. Specifically, 1 μl/μl of each of apo-clytin-II expressionvector and the internal standard firefly luciferase expression vectorand 6 μl FuGene HD are added into 100 μl of the DMEM medium, and themixture is placed at room temperature for 15 min. 50 μl of solution ofthe DNA-FuGene complex is added to the cells in the six wells. After24-h of culturing, the cells are washed with 2 ml of cold PBS for threetimes and 250 μl of cold PBS is added into the cells to recover thecells. The recovered cells are disintegrated with an ultrasonichomogenizer as an enzyme solution used for determination.

Example 4 Determination of Luminescent Activity in Cultured Animal Cells

(1) Determination with Firefly Luciferase as Internal Standard

10 μl of the enzyme solution obtained in Example 3 is added into 50 μlof the enzyme assay reagent (Promega Company) to initiate the luminousreaction. The luminescent activity is determined by a luminescencemeasuring device (AB2200, manufactured by Atto Company) for 10 secondsand expressed in relative luminescence intensity.

(2) Determination of Luminescent Activity

50 μl of the enzyme solution obtained in Example 3 is added into 950 μlof 50 mM Tris-HCl (pH 7.6) containing 1 μl of mercaptoethanol (WakoChemicals), 1 μl of coelenterazine (manufactured by Chisso Company), and10 mM of EDTA (Wako Chemicals), and the resulting mixture is placed at4° C. for 3 h for the regeneration of clytin-II to occur. 50 mM Tris-HCl(pH 7.6) solution containing 100 μl of 50 mM CaCl₂ is added into 10 μlof the regenerated solution in order for the regenerated solution toemit light. The light emission is determined by a luminescence measuringdevice (AB2200, manufactured by Atto Company) for 10 seconds, and themaximum luminescence intensity is expressed by relative luminescencevalue.

The results are summarized in Table 3. The activation rate is calculatedaccording to the activity of firefly luciferase used as the internalstandard. According to the results, the codon-optimized clytin-IIprotein of the present invention exhibits an activity of about 15 timesor more, compared with the wild-type apo-clytin-II.

TABLE 3 Comparison of expression of the wild-type clytin-II gene and thehuman-type clytin-II gene in cultured cells Clytin-II Clytin-II Fireflyluminescent luminescent luciferase activity/firefly activity activityluciferase activity Activation Used plasmid (rlu) (rlu) (×1000) rate —24 100 — — pCMX-CLII + 1504 255724 5.8 1.0 pGL-control pCMX-hCLII +21892 187976 116.4 15.8 pGL-control

Example 5 Construction of Codon-Optimized clytin-II Protein ExpressionVector in E. coli

In order to express the codon-optimized apo-clytin-II nucleic acid in E.coli, the expression vector piP-H-L(6) is constructed as the startingmaterial. Specifically, the linker having a chemical synthesizedmultiple cloning sequence H/P/S/Xb/Xh/B:Linker-F (5′ AG CTT CTG CAG GTCGAC TCT AGA CTC GAG G 3′) (SEQ ID NO: 9) and H/P/S/Xb/Xh/B:Linker-R (5′GA TCC CTC GAG TCT AGA GTC GAC CTG CAG A 3′) (SEQ ID NO: 10) is insertedat the HindIII-BamHI site of the piP-(His6)HE vector, as described inJapanese Patent Publication 2008-22848, to construct piP-H-L(6). Thebasic vector piP-H-L(6) is controlled by the E. coli lipoproteinpromoter and the lactose operon, which has an OmpA sequence forsecretion, a six histidine sequence for purification with chelate gel,and various multiple cloning sequences.

The construction of the codon-optimized apo-clytin-II protein expressionvector using the basic vector piP-H-L(6) is as follows. With pBlue-hCLIIas template, PCR is performed by using the PCR primer pair:hCLII-1N/HindIII (5′ ggc aag ctt GTG AAG CTG GAC CCC GAC TTC 3′) (SEQ IDNO: 11) and hCLII-2C/XhoI (5′ ggc CTC GAG TTA GGG GAC GAA GTT GCC GTA3′) (SEQ ID NO: 12) with a PCR kit (manufactured by Nippon Gene Company)(cycling conditions: 25 cycles; 1 min/94° C., 1 min/50° C., 1 min/72°C.). The obtained segment is purified with a PCR purification kit(manufactured by Qiagen Company), digested with the restriction enzymeHindIII/XhoI, and then inserted into the restriction enzyme HindIII/XhoIsite of piP-H-L(6) to construct the expression vector piP-H-hCLII asshown in FIG. 2. Further, the base sequence is determined with a DNAsequencer (manufactured by Applied Biosystems Company, ABI) to confirmthe inserted DNA.

Example 6 Expression of Codon-Optimized apo-clytin-II Protein in E. Coli

In order to express the codon-optimized apo-clytin-II protein(hereinafter sometimes called “hCLII”) in E. coli, the recombinantplasmid piP-H-hCLII fabricated in Example 2 is used. The recombinantplasmid piP-H-hCLII is introduced into the E. coli strain WA802 byroutine methods, and the obtained transformant is implanted into a 10 mlLuria-Bertani (LB) liquid medium (10 g bacto tryptone, 5 g yeastextract, and 5 g NaCl contained in each liter water, pH 7.2) containingampicillin (50 μg/ml), and cultured at 30° C. for 18 h. Then, theculture is added into a new LB liquid medium 400 ml×5 (total amount 2L), and cultured at 30° C. for 18 h. After culturing, the bacteria arecentrifuged for recovery (5,000 rpm, 5 min), and the cultured bacteriaafter collection is used as a starting material for protein extraction.

Example 7 Extraction and Purification of Codon-Optimized apo-clytin-IIProtein from Cultured Bacteria

The cultured bacteria after collection is suspended in 200 ml of 50 mMTris-HCl (pH 7.6), cooled in an ice bath, and subjected to an ultrasonictreatment (manufactured by Branson Company, Sonifier model cycle 250)for 3 min for 3 times, and the disintegrated bacteria liquid iscentrifuged at 10,000 rpm (12,000×g) at 4° C. for 20 min. The obtainedsoluble fraction is supplied to a nickel-chelated column (Amershambiosciences Company, column size: diameter 2.5 cm×6.5 cm) balanced by 50mM Tris-HCl (pH 7.6), to allow the codon-optimized apo-clytin-II proteinto be adsorbed. After being washed with 500 ml of 50 mM Tris-HCl (pH7.6), the codon-optimized apo-clytin-II protein is eluted with 0.1 Mimidazole (manufactured by Wako Chemicals Company). With bovine serumalbumin (manufactured by Pias Company) as sample, the concentration ofthe purified protein is determined by a commercially available kit(manufactured by Bio-Rad Company) according to the Bradford method. As aresult, 45.7 mg codon-optimized apo-clytin-II protein is obtained from 2L of cultured bacteria. The purification is determined to be 95% or moreby the SDS-PAGE analysis.

Example 8 Determination of Luminescent Activity

The light emission of clytin-II during purification is determined in thefollowing manner, wherein clytin-II is constructed with codon-optimizedapo-clytin-II protein as a component. After 2-mercaptoethanol (1 μl) andsubstrate coelenterazine (1 μg/μl) dissolved in ethanol are blended into990 μl of 30 mM Tris-HCl (pH 7.6) containing 10 mM EDTA, 10 μl ofhuman-type apo-clytin-II solution is added, and placed on ice (4° C.)for 2 h. 100 μl of 50 mM calcium solution is added into 10 μl ofregenerated human-type clytin-II solution to initiate the luminousreaction. The luminescent activity is determined with a luminescencemeasuring device (AB2200, manufactured by Atto Company) by determiningthe light emission for 10 seconds. The maximum luminescence intensity isexpressed by the relative luminescence value (Table 4).

TABLE 4 Purification yield of codon-optimized apo-clytin-II proteinTotal Total amount Total Specific amount of protein activity activityRecovery rate (%) Stage (ml) (mg) (×10⁹rlu) (×10⁹/mg) Protein ActivityCrude extract 200 720 89.3 1.2 100 100 Nickel chelate 30 45.7 59.2 27.86.3 66 gel

INDUSTRIAL APPLICABILITY

The codon-optimized nucleic acid for coding apo-clytin-II is introducedinto a host cell to get the codon-optimized apo-clytin-II protein. Whenforming the complex consisting of the codon-optimized apo-clytin-IIprotein, the luminescent substrate and molecular oxygen, thecodon-optimized apo-clytin-II protein exhibits a more significantluminescent activity than that using the wild-type apo-clytin-II.Furthermore, the clytin-II fabricated by the present invention is veryeffective in various applications, such as the detection ofintracellular calcium change.

SEQUENCE LISTING

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure of the presentinvention without departing from the scope or spirit of the invention.In view of the foregoing, it is intended that the present inventioncover modifications and variations of this invention provided they fallwithin the scope of the following claims and their equivalents.

1. A codon-optimized nucleic acid, for coding apo-clytin-II protein,wherein the codon-optimized nucleic acid is SEQ ID NO:3 expressed by anexpression vector/host systems comprising mammalian cells, bacteriacells, or yeast cells.
 2. A codon-optimized nucleic acid, formed byfusing the codon-optimized nucleic acid according to claim 1 with anucleic acid that codes other proteins.
 3. An expression vector,comprising a codon-optimized nucleic acid for coding an apo-clytin-IIprotein wherein said codon optimized nucleic acid is SEQ ID NO:3 andwherein said codon-optimized nucleic acid is under a regulatory controlof a promoter that functions in mammalian cells and is expressed by anexpression vector/host systems selected from the group comprisingmammalian cells, bacteria cells, or yeast cells.
 4. The expressionvector according to claim 3, comprising the codon-optimized nucleic acidfor coding the apo-clytin-II protein, and a regulatory sequence forcontrolling an expression of the codon-optimized nucleic acid for codingthe apo-clytin-II protein in the mammalian cells.
 5. A recombinant hostcell selected from the group comprising mammalian cells, bacteria cellsor yeast cells, wherein said recombinant host cell comprises acodon-optimized nucleic acid according to claim 1 for coding anapo-clytin-II protein.